重组囊素肽的构建表达及其对禽流感灭活苗免疫效果的影响

Effect of recombinant bursin peptides on immunomodulatory to avian influenza vaccine in chickens

为重组表达不同拷贝串联囊素(BS)活性肽及评价其对禽流感灭活苗的免疫效果,本研究以囊素五肽和三肽的氨基酸序列为基本单元,设计了3种不同串联方式的囊素肽,按照大肠杆菌偏爱密码子设计其引物,利用SOE-PCR技术扩增了3种囊素肽编码基因,分别克隆至p ET-32a中,构建重组质粒p ET-BS1、p ET-BS2和p ET-BS3,并转化于大肠杆菌Origami TM B中诱导表达。SDS-PAGE和western blot结果表明,3种重组囊素肽BS1、BS2和BS3均以可溶形式表达,与天然囊素肽具有相似的免疫原性。将3种重组囊素肽分别与禽流感灭活病毒联合免疫雏鸡,免疫效果检测表明,与单独免疫灭活病毒组相比,3种重组囊素肽均能够明显促进鸡血清中抗体产生,提高外周血淋巴细胞增殖能力;但仅BS1能够同时提高血清中IL-4和IFN-γ的含量。本研究通过原核表达制备了3种具有良好免疫活性的重组囊素肽,为其作为免疫增强剂提供了实验依据。

To evaluate the immunopotentiating bursa peptides, three fusion tandem peptides encoding sequences containing Bursin （BS） or Bursopentin （BP5） in different combinations were synthesized according to the codon usage bias of E.coli and amplified by SOE-PCR, which were insert into pET-32a to construct 3 recombinant plasmids for expressing in E.coli, respectively. Then the 3 fusion proteins of BS1, BS2 and BS3 were highly expressed with MW about 21 ku in a soluble form under induction induced of IPTG at 22℃. In addition, a total of 120 one-day-old chickens were divided into different groups and intramuscularly co-immunized with killed avian influenza virus （AIV） and purified protein BS~, BS：, BS3, BS and BP5, respectively. The humoral and cellular immune responses was examined in serum of the chickens, and the data showed that compared to the control group immunized with killed AIV, the level of antibody titer and ability of T lymphocyte proliferation in groups co-immunized with killed AIV and BS,, BS： or BS3 were significantly higher, while only the group co-immunized with killed AIV and BS1 improve both level of IL-4 and that of IFN-γ. These results would be helpful for using prokaryotic expressed bursal peptides as an adjuvant for vaccines.